Construction of prokaryotic expression vectors pGEX-6p-1-aroA J. sp and pGEX-6p-1-aroA E. coli

The oligonucleotide primer sequences were as follows: primer 1 (5′-CGCGGATCC ATGACCAGTCCTGATTGGCATGC-3′) (the BamHI site is underlined); primer 2 (5′-CCGGAATTCTCAGCCCTCCGACGCCTCG-3′) (the EcoRI site is underlined), which was designed according to the sequence derived from the insert of plasmid pZY3 and supplied by the Genescript; primer 3 (5′-CGCGGATCCATGGAA TCCCTGACGTTACAACCCATCGCTCGTG-3′); and primer 4 (5′-CCGGAATTC TCAGGCTGCCTGGCTAATCCGCGCCAGCT-3′), which was specific for amplifying aroA_{E. coli} gene and was designed based on the sequence available in GenBank (GenBank:{"type":"entrez-nucleotide","attrs":{"text":"X00557","term_id":"40965","term_text":"X00557"}}X00557). The aroA_{J. sp} and aroA_{E. coli} genes were amplified, using the recombinant plasmid pZY3 and the genomic DNA from E. coli as the template, respectively. The FastPfu DNA polymerase (Transgen, Beijing, China) was used for all the reactions under the following conditions: 30 cycles of 94 °Cfor 20 sec, 55 °C for 20 sec, and 72 °C for 1 min. After PCR, the amplified products were gel-purified, digested with BamHI and EcoRI, and ligated into the pGEX-6p-1 vector.